The chromosomal DNA of a eukaryote forms a complex with protein, and such a complex is called chromatin. More specifically, chromatin involves repeated structures of nucleosomes connected spirally; the nucleosome assumes a conformation in which DNA of 146 base pairs is wrapped 1.75 times around a histone core (a histone octamer) containing 2 molecules each of 4 types of histone proteins: H2A, H2B, H3, and H4. The binding between DNA and histone inhibitorily acts on transcription. It is known that the nucleosome is loosened with histones dissociated in a chromosome containing a gene locus whose transcription is active. Histone consists of a globular carboxyl terminus and a linear amino terminus (histone tail); the lysine residue and asparagine residue of the histone tail are known to undergo various modifications such as acetylation, methylation, phosphorylation, and sumoylation. Outlines of acetylation and methylation among protein post-translational modifications of the lysine residue of histone are shown in FIG. 1. As shown in FIG. 1, the degree of acetylation consists of one level (acetylated lysine (ε-N-acetyllysine)) for acetylation, while the degree of methylation consists generally of 3 levels (monomethylated lysine (ε-N-methyllysine), dimethylated lysine (ε-N,N-dimethyllysine), and trimethylated lysine (ε-N,N,N-trimethyllysine))) for methylation.
The methylation modification is known to cause transcriptional control, silencing, chromatin condensation, and the like. The methylation of histone is induced by histone methyltransferase (HMT). FIG. 2 shows the names of human histone methyltransferases heretofore known, the lysine sites at which these enzymes are methylated (lysine sites), and the influence of the methylation of the lysine sites on transcription (transcriptional enhancement or transcriptional repression). The histone methylation is known to be associated with various diseases. For example, Non-Patent Document 1 describes that various histone methyltransferases such as SUV39H1, EZH2, MLL, NSD1, and RIZ are responsible for tumor development. Non-Patent Documents 2 and 3 also describe that the methylation of histone H3K9 and histone H3K27 is observed together with increased DNA methylation and decreased histone acetylation in the promoter region of a cancer suppressor gene whose expression is suppressed in cancer cells. In addition, Non-Patent Document 4 describes that increased methylation of histone H4K20 is observed in common to many cancers. Non-Patent Document 5 also describes that the formation of a heterochromatin induced by the methylation of histone is involved in neurodegenerative diseases such as myotonic dystrophy and Friedreich motor ataxia. Non-Patent Document 6 also describes that iPS cells were induced by adding BayK8644 as an agonist of L-type calcium channels and BIX-01294 as an inhibitor for the histone methyltransferase G9a to Oct4/Klf2 and gene-introduced mouse embryo fibroblasts.
Thus, a histone methyltransferase inhibitor is expected as a therapeutic agent for diseases such as cancer and neurodegenerative disease and to be applied to regenerative medicine using iPS cells. Accordingly, screening for histone methyltransferases is attempted. For example, Non-Patent Document 7 describes a method which involves mixing [methyl-3H]-SAM as S-adenosylmethionine (S-(5′-adenosyl)-L-methionine: SAM) whose methyl group is labeled with tritium, a peptide consisting an amino acid sequence of amino acid 1 to 19 of histone H3 (histone H3 (1-19) peptide), and a histone methyltransferase for reaction, followed by measuring the radioactivity of the histone H3 (1-19) peptide to measure the amount of the methyl group transferred to lysine to thereby measure the histone methylation activity of the histone methyltransferase (so-called RI method) (FIG. 3). Non-Patent Document 8 also describes a method for screening for histone methyltransferase inhibitors by an Elisa method (FIG. 4). However, the RI method has had problems of being limited in safety because of using the radioisotope, being complicated because of requiring relatively many experimental procedures including the adsorption of a reaction product to filter paper and the washing of the filter paper, and the like (FIG. 5). As shown in FIG. 5, the Elisa method has had problems of an extremely large number of experimental procedures such as a washing procedure and a large amount of time required. Under such circumstances, there has been a need for an evaluation system for histone methylation activity, wherein the system has no problem with safety and is simple in the experimental procedures and short in time required.
A known simple method for measuring histone deacetylase activity includes a method using a substrate peptide represented by X-X-Lys(Ac)-(dye) (that is, a substrate peptide represented by X-X-(Ac)Lys-(dye) in Patent Document 1), (wherein X represents any amino acid residue; Lys(Ac) represents a lysine residue whose ε amino group (an amino group at the ε position) is acetylated; and (dye) represents a dye label bound to the lysine residue) (see Patent Document 1). This method uses the property that the cleavage activity of a certain peptidase remains lowered in a state where the substrate peptide remains acetylated, while the cleavage activity of the peptidase is increased when the substrate peptide is deacetylated.
With regard to methylation at the ε position of lysine, Non-Patent Documents 9 and 10 describe that methylation at the ε position of lysine residue of a peptide renders the peptide less susceptible to decomposition by trypsin than no such methylation. However, it has been uncertain whether the methylation at the ε position of lysine can actually be used for a method for measuring histone methyltransferase activity, for example, because it consists of 3 steps (monomethylation, dimethylation, and trimethylation), unlike the acetylation, and decreases the cleavage activity of a certain peptidase as the reaction proceeds, contrary to the deacetylation in the above-described Patent Document 1.